Fiber deprivation and microbiome-borne curli shift gut bacterial populations and accelerate disease in a mouse model of Parkinson’s disease

Summary Parkinson’s disease (PD) is a neurological disorder characterized by motor dysfunction, dopaminergic neuron loss, and alpha-synuclein (αSyn) inclusions. Many PD risk factors are known, but those affecting disease progression are not. Lifestyle and microbial dysbiosis are candidates in this context. Diet-driven gut dysbiosis and reduced barrier function may increase exposure of enteric neurons to toxins. Here, we study whether fiber deprivation and exposure to bacterial curli, a protein cross-seeding with αSyn, individually or together, exacerbate disease in the enteric and central nervous systems of a transgenic PD mouse model. We analyze the gut microbiome, motor behavior, and gastrointestinal and brain pathologies. We find that diet and bacterial curli alter the microbiome and exacerbate motor performance, as well as intestinal and brain pathologies, but to different extents. Our results shed important insights on how diet and microbiome-borne insults modulate PD progression via the gut-brain axis and have implications for lifestyle management of PD.

kristopher.john.schmit@med. uni-tuebingen.de (K.J.S.), paul.wilmes@uni.lu (P.W.) In brief Schmit et al. investigate how dietary fiber deprivation in combination with a bacterial toxin affect PD progression. They point to a translational PD-relevant sequence of events exacerbating disease progression in a disease mouse model and put forth the idea of lifestyle adaptations to mitigate the disease.

INTRODUCTION
Lifestyle and environmental factors contribute to chronic neurodegenerative diseases, burdening socio-economic structures in an ever-aging population. 1,2 The incidence of Parkinson's disease (PD), the second most common neurodegenerative disease, has increased over the last decades 3 and will increase further. 4 Only 5%-10% of cases are familial. 5 Because of the protracted nature of this disease, years lived with disabilities pose a significant burden on patients, their families, and society. 6 Genetic predispositions and environmental/lifestyle factors contribute to PD onset and progression. 7 Such disease-promoting factors are exposure to chemicals (e.g., pesticides), head trauma, physical activity, stress, and diet. 8 Which factors modulate disease progression has only recently been investigated. 9 Increasing evidence has highlighted the importance of diet in PD progression. A fiber-rich Mediterranean diet (fresh fruits and vegetables) reduces the risk for PD. 10 Conversely, a ''Western'' diet, with low amounts of fiber and high amounts of (E. coli) and Salmonella typhimurium, which both express curli, a major biofilm component. 20 Curli is an amyloidogenic protein that has similarities to b-amyloid and a-synuclein (aSyn), 25,26 and it cross-seeds with aSyn. 27,28 aSyn aggregates are present in the gut of patients with PD. 29-32 Braak and colleagues proposed that aSyn aggregates in the enteric nervous system (ENS) precede those in the lower brainstem of the central nervous system (CNS), and that aSyn propagates retrogradely in a ''prion-like'' manner, via the vagus nerve, to and throughout the brain. 33 Because environmental factors modulating disease act in concert, we investigated the effect of dietary fiber deprivation and bacterial curli exposure, individually or combined, on a human aSyn-overexpressing transgenic mouse. Our treatment protocol primed the naive untreated microbiome with a ''Westernized'' fiber-deprived diet, 16 followed by exposure to curli-producing bacteria. 27 We analyzed the mice at gut microbial, behavioral, gastrointestinal, and neuropathological levels. Overall, transgenic, but not wild-type mice, were susceptible to the different challenges. In transgenic mice, we found that, while aSyn overexpression was largely responsible for motor impairments, fiber deprivation caused dysbiosis, with increased levels of Akkermansia spp. and Bacteroides spp., sparking mucus erosion, facilitating entry of enteric pathogens. 16 This resulted in increased PD-like pathologies, including increased motor dysfunction, aSyn aggregation in the ENS and CNS, and nigrostriatal degeneration. We believe that our study sheds light on how a combination of internal and external pathogenic factors drives PD-like pathologies in the CNS and ENS. Our findings may have important implications for lifestyle adjustments that could mitigate PD progression.

RESULTS
Thy1-Syn14 mice overexpressed aSyn in brain and gut with regional differences The model we used overexpresses human wild-type aSyn under the transcriptional control of the Thy1 promotor, and it carries 13 copies of the transgene. 34 Only protein levels in bulk brain tissue have been reported. 34 Thus, we first determined baseline gene expression and protein levels of aSyn in the nigrostriatal pathway and in the colon of naive 9-month-old mice. We hypothesized that, at this age, the mice would already have developed PDlike pathology (e.g., behavioral deficits, described below). In our mice, mRNA level for the murine aSyn (Snca) gene (Figure 1A, left panel) did not differ between genotypes, but it was significantly (p < 0.0001) higher in the dorsal striatum versus the ventral midbrain. For the human aSyn transgene (SNCA) (Figure 1A, right panel), we only detected a signal in transgenic (TG) mice, and those levels did not differ between the two regions. When we stained for total aSyn, using an antibody ( Figure S1A) that detects both murine and human aSyn, we saw that aSyn was present in both wild-type (WT) and TG mice in the dorsal striatum as well as in the substantia nigra pars compacta (SNpc). By western blot with that same antibody, we observed a 2.91-fold increase (p = 6.67EÀ4) in the dorsal striatum and a 6.67-fold increase (p = 6.67EÀ4) in the ventral midbrain of TG compared to WT mice ( Figures 1B and S1B). Additionally, aSyn protein levels were significantly higher in the dorsal striatum compared to the ventral midbrain (WT: p = 1.52EÀ2; TG: p = 1.55EÀ4; Figures 1B and S1B).
In the colon, which we split into proximal and distal parts, we observed that the mRNA levels for Snca and SNCA were much lower compared to those in the CNS ( Figure 1C). A possible explanation could be the lower neuronal density in colon compared to brain. In the colon, only about 1% of all cells are neurons, 35 whereas ventral structures of the midbrain (e.g., SNpc and ventral tegmental area) have an estimated 15%-20% neurons. 36-39 Nevertheless, we observed that again SNCA was only expressed in TG mice, and Snca was expressed at significantly (p < 0.0001) different levels between proximal and distal colonic regions but not between genotypes (Figure 1C). Additionally, we stained for human aSyn and total aSyn ( Figure 1D). The latter was expressed in mice of both genotypes, while only TG mice expressed human aSyn ( Figure 1D, left bottom panel).
Based on our analysis, the Thy1-Syn14 model is an appropriate system to investigate the effect of environmental challenges delivered through the gut in the context of aSyn overexpression.
Thy1-Syn14 mice exhibited progressive motor deficits PD-like motor deficits were reported in different mouse models of the disease. 40 We assessed grip strength, hindlimb reflexes, and coordination/movement as measures of motor dysfunction in naive cohorts of 3-, 6-, and 13-month (M)-old mice. For hindlimb reflexes and coordination/movement assessments, we added the baseline results from our treatment cohort at 9 months of age (see below).
Finally, we tested mice for fine motor skills with the adhesive removal test. We did not observe relevant motor deficits in young mice (3M and 6M; Figure 1G). At 9 and 13 months, we saw that both sensitivity (time at touch, left panel) as well as coordination (time at removal, right panel) were significantly delayed in TG mice (time at touch -9M: p = 7.57EÀ7, 13M: p = 0.057; time at Removal -3M: p = 0.016, 9M: p = 1.12EÀ6, 13M: p = 0.016).
Taken together, these data indicate that overexpression of human aSyn drives progressive motor dysfunction in Thy1-Syn14 mice.
Fiber deprivation induced PD-related microbiome changes Onset and progression of PD have been linked to environmental factors, 41-44 of which some, e.g., diet, impact the gut microbiome. 45,46 Changes in gut microbial composition have been described in patients with PD 47-53 and in animal models. [54][55][56] It is unknown how these changes affect the progression of PD. Using 16S rRNA gene amplicon sequencing, our goal was to first understand how the exogenous challenges affected, independently or in combination, the gut microbiome in our mice. The exogenous challenges were as follows: (1 ) Diet: a normal fiber-rich (FR) or a fiber-deprived (FD) diet (2 ) Gavages: PBS, curli knockout isogenic E. coli (DEC), or curli-producing E. coli (EC) We started by looking at how the challenges affected microbial diversity. We saw that the FD diet and TG challenges both reduced inner-group diversity (alpha diversity) but that the gavage challenges, PBS or E. coli (EC or DEC) ( Figure S2A), did not. Alpha diversity was lowest at week 2 (WT: p = 0.045; TG: p = 6.1EÀ4) and week 9 (TG: p = 9.7EÀ3) in FD-challenged and more strongly so in TG mice (Figure 2A). We did not observe this microbial shift at weeks 5 and 6. A possible explanation is that the initial treatment with E. coli at week 2 caused a transitory disruption in the microbial balance. We made similar observations for beta diversity, where the diet challenge was the main driver (adonis p = 0.001) of dissimilarities between groups (Figure S2B, middle panel). However, in contrast to alpha diversity, the gavage challenges, PBS or E. coli (EC or DEC), also signifi-cantly (adonis p = 0.001) contributed to microbial community shifts ( Figure S2B, right panel). This might have been due to the PBS-gavaged mice having all been FD challenged. Hence, solely the FD challenge drove the observed rapid shift in microbiome ( Figure 2B). Already at week 2, the FD and FR groups, independent of the other challenges, formed two homogeneous clusters, which, unlike the alpha diversity results, lasted until the end of the in-life phase. Such a shift is indicative of dysbiosis. Changes in the Firmicutes to Bacteroidetes ratio, formerly a standard for microbial imbalance, 57,58 showed significantly higher levels of Firmicutes compared to Bacteroidetes in FD-challenged mice at the end of the in-life phase (week 9, Figure S3). Abundance differences on the genus level were again driven though the FD diet challenge ( Figure S4A). When we focused on our most relevant genera ( Figure 2C), we observed that the FD challenge led to higher levels in Intestinimonas, Odoribacter, Colidextribacter, Rikenellaceae RC9 gut group and Akkermansia and decreased levels in Lachnospiraceae NK4A136 group and Lactobacillus over time ( Figures 2C and S4A). Other taxa presented with a more fluctuating behavior over time (Figures 2C and S4A). When compared to data from patients with PD (Table 3 in Boertien et al. 47 ), FD-challenged mice showed similar changes in Akkermansia, Lachnospiraceae, Roseburia, and Prevotellaceae, while Lactobacillaceae and its genus Lactobacillus were inversely altered (Table S1).
When we checked for the relative abundance of E. coli, the species we gavaged with, we did not detect it in our 16S rRNA gene amplicon sequencing data (not shown). We performed a probe-based quantitative real-time PCR analysis, revealing that fecal E. coli levels were significantly (Kruskal-Wallis, p = 4.55EÀ12) increased in FD-diet-challenged mice compared to mice on the FR diet ( Figure S4B), as well as being significantly (Mann-Whitney U test, false discovery rate [FDR] = 0.027) higher in mice gavaged with PBS compared to EC-challenged mice ( Figure S4C) (independent of genotype and time points). A possible explanation for higher fecal E. coli levels in FD-diet-fed mice could be the inability of E. coli to implant in the eroded outer mucus layer but still sufficiently so as to have pathogenic effects (see below). Looking into how levels changed over time, starting when we first gavaged animals (week 2), we did see significantly higher levels of E. coli in FD-diet-fed compared to FR-diet-fed mice (Kruskal-Wallis; WT: week 5, p = 0.0366; TG: week 2, p = 0.00793; week 5, p = 0.0267; Figure S4D), but we did not observe incrementally increasing fecal E. coli levels. Similarly, in another study, 27 E. coli administration induced PD-like pathologies without colonizing the gut.
In summary, the FD challenge caused reduced gut microbial diversity with changes in taxa associated with gut health and disease. Of note were reduced levels of Lactobacillus, a probiotic genus, 59,60 reported to have neuroprotective 61 and gut-barrierintegrity-enhancing functions, 62 and of the Lachnospiraceae NK4A136 group, which inversely correlates with risk for PD or dementia. 63 Additionally, Lachnospiraceae NK4A136 group and Roseburia are important butyrate producers, promoting proper gut barrier function. 64 Together with higher levels of mucin-foraging genera Akkermansia, Alistipes, and Bacteroides, 16,65,66 the passage of pathogenic factors through the barrier might be increased.
Microbiome-driven colonic mucus erosion in fiberdeprived Thy1-Syn14 mice The mucus layers of the colon have two basic functions: the inner layer acts as a physical barrier to prevent pathogens from reaching the gut epithelium, and the outer layer harbors commensal bacteria interacting with the host. 22 Increasing levels of mucindegrading bacteria, such as A. muciniphila and certain Alistipes spp. and Bacteroides spp., 16 cause mucus thinning and thus enable epithelial access for pathogens. In a pilot study ( Figures S5A-S5C), where we fed an FD diet to TG mice and WT mice, we saw that, after 1 week, there was a rapid and significant (FDR = 0.004; Figure S5C) erosion of the outer mucus layer in FD-diet-fed mice, independent of their genotype. After 3 weeks, the outer mucus layer remained significantly (FDR: 0.036; Figure S5C) thinned in mice of both genotypes. The inner mucus layer did not differ between the different conditions (Figure S5B). This initial observation showed how rapidly the outer mucus erodes upon fiber deprivation.
In FD-challenged mice, similar to the pilot study, we did not detect changes of the inner mucus layer ( Figures S5D and  S5E). Our results for the outer mucus layer on the other hand clearly showed that the FD challenge caused significant (p = 1.15EÀ12) thinning of the outer mucus layer ( Figure 3A). The layer thickness decreased extensively by 49.1%-92.9% in the FD diet group ( Figure 3B). Hence, the habitat for specific gut bacteria was reduced, which we expected to directly affect microbial diversity. Therefore, we compared alpha diversity and mucus thickness using the Spearman's rank test ( Figure S5F). We were specifically interested in dietary-or transgene-driven associations. While we only saw moderate dietary-driven correlations (FR: r = À0.47, p = 0.051; FD: r = 0.38, p = 0.087; Figures S5G and S5H), there was a significant positive correlation between alpha diversity and mucus thickness in TG mice ( Figure 3C), indicating that the outer mucus was more susceptible to FD-induced thinning when aSyn was overexpressed.
Curli exposure and fiber deprivation disrupted gut barrier integrity in Thy1-Syn14 mice Our microbiome data indicated changes in gut-barrier-integrityrelevant taxa. To assess gut barrier integrity, we looked at two different tight junction proteins, zonula occludens-1 (ZO-1) and occluding, by western blot. These tight junction proteins, form, with others, a continuous barrier between epithelial cells that is disrupted by pathological conditions, 67-69 resulting in a ''leaky gut,'' which has been described in patients with PD and other diseases. 70,71 Interaction of genotype and diet was the main driver of differences in ZO-1, which not quite reached significance (p = 0.0698, by permutational multivariate analysis of variance). When we investigated group differences for each genotype, we saw that the FD EC challenge in TG mice had overall the lowest levels of ZO-1, with notable (Mann-Whitney U test, before correction; p = 0.03) lower levels than the FD DEC-challenged TG mice ( Figures 3D and 3E).
For occludin, we detected a band at 55 kDa (up to 65 kDa; referred to as 55 kDa here) and another band at 25 kDa in our gels ( Figure 3G). This smaller isoform is most likely due to alternative gene splicing 72,73 and has been observed in another study. 74 The 25-kDa isoform levels were significantly higher in both WT (Kruskal-Wallis, p = 0.00335) and TG (Kruskal-Wallis, p = 0.00483) mice compared to the 55-kDa form. We analyzed the datasets for the 55-kDa and the 25-kDa occludin isoforms separately ( Figure 3F). For both, the genotype did not explain group differences. Thus, we analyzed each genotype separately. While in WT mice, occludin levels were quite variable, in TG mice, we saw significantly (Mann-Whitney U test before correction for multi-comparison) lower levels of 55-kDa (p = 0.022) and 25-kDa (p = 0.035) occludin, respectively, after the FD EC compared to the FD DEC challenges ( Figure 3F). Thus, the level of occludin was lowered by all treatments in TG mice compared to the FD PBS control group, indicating that occludin is a sensitive marker for gut barrier dysfunction ( Figure 3F).
We can conclude that the EC challenge alone affects the gut barrier integrity, but the effect is most pronounced when combined with the transgene and the fiber deprivation challenges. Bacterial curli increased pS129-aSyn-positive structures in the colonic myenteric plexus of fiberdeprived Thy1-Syn14 mice aSyn aggregation in the gut is detected in patients with PD. 33,75,76 To test for aSyn aggregation in the gut of our mice, we used an antibody directed against phosphorylated Ser129 aSyn (pS129-aSyn). This type of antibody is commonly used to detect aSyn aggregation in murine and human CNS 77 and ENS. 78,79 We quantified pS129-aSyn-positive deposits in ganglions of the myenteric plexus positive for protein gene product 9.5 (PGP9.5), a neuronal cytoplasmic marker. 80 Other pS129-aSyn-positive structures in the submucosal plexus and submucosa were irregular and mainly detected in TG mice. This, however, did not differ between the different treatment groups, and we could not determine the cell types involved.
First, the staining showed that both WT and TG mice had pS129-aSyn-positive signals in ganglions of the myenteric  Table S2. plexus (micrographs in Figure 4A). Quantification of these signals showed that only TG mice exposed to the combined challenges of FD and curli-expressing E. coli (FD EC) had increased levels of pS129-aSyn in PGP9.5-positive ganglions (vs. TG FD PBS: p = 0.019; vs. WT FR EC: p = 0.019; vs. TG FR EC: p = 0.008; vs. WT FD DEC: p = 0.019; Figure 4B).
In summary, the strongest increase of pS129-aSyn-positive structures occurred in the myenteric plexus of the colon of TG EC FD-challenged mice.
Bacterial curli and dietary fiber deprivation combination exacerbated motor deficits in Thy1-Syn14 mice At baseline, in 9-month-old TG mice, we did already observe motor deficits. Therefore, we examined if the challenges, individually or in combination, exacerbated these deficits.
Three different tests were chosen to assess motor performance: hindlimb clasping, grip strength, and adhesive removal. At baseline, TG mice differed significantly from WT littermates in the first two tests by showing age-related decline in performance ( Figures 1F, S6A, and S6B). These declines were not affected by the other challenges and thus most likely driven by aSyn overexpression.
The adhesive removal test was used to assess fine motor function. The test consists of measuring the latencies of touch and removal, which at baseline were significantly (p < 0.0001) greater in TG mice compared to WT littermates ( Figure 1G). This was still the case at the end of the 9-week challenge phase (p < 0.0001; Figures 5A and S6C). Hence, aSyn overexpression was again key in driving motor impairment. Additionally, EC-challenged TG mice, independent of diet, showed increased latencies of touch and removal compared to WT mice ( Figures 5A and S6C). Further, we saw a noticeable increase in both measures for a subset of TG FD EC-challenged mice. To understand how the ability to remove the adhesive tape changed over time, we (1) subtracted the time of touch from the time of removal and then (2) compared ''baseline'' to ''endpoint'' results. We found that, in the TG FD EC group, three out of six mice had reduced coordinative ability to remove the adhesive tape compared to their initial performance ( Figure  5B). Thus, after grouping mice into categories, our results indicate that the combination of the FD diet and curli exacerbated the motor phenotype in a subset of TG mice.
When we compared these results to our baseline assessment of 13-month-old untreated transgenic mice, there was no significant difference. We believe that this could indicate that severity of motor deficits may already have plateaued. They may be due to expression of aSyn in the spinal cord as has been described in models using the Thy1 promotor. 81 Only the further increase picked up in the sensitive adhesive removal test after FD and EC challenge may be due to the loss of striatal TH-positive axons and nigral TH-positive neurons (see below).
Bacterial curli increased pS129-aSyn-positive structures in the nigrostriatal pathway of Thy1-Syn14 mice All neuropathological analyzes were limited to TG mice, since they had pre-existing PD pathologies and were susceptible to the challenges. In a first step, we determined abnormal aSyn structures in the nigrostriatal pathway. We quantified pS129-aSyn-positive structures in the dorsal striatum and the SNpc. We observed that the EC challenge was the main driver of enhanced pS129-aSyn aggregation in both regions ( Figure 6).
In the dorsal striatum, we measured significantly (p = 0.018) higher levels of pS129-aSyn-positive structures in FR EC-challenged TG mice compared to the FR DEC group ( Figure 6A Figure 6B, top panel). Qualitatively, pS129-aSyn-positive structures observed in cell bodies appeared diffuse in the cytoplasm but more compact in the nuclei ( Figure 6B, bottom panel, white arrowhead). Importantly, nuclear aSyn aggregation has been demonstrated in brains of patients with Lewy Boyd dementia. 82 However, in FD EC-challenged mice, we also found dense pS129-aSyn-positive deposits in cell bodies ( Figure 6B, bottom panel, blue arrowheads). All other pS129-aSyn-positive structures that were not within cell bodies were of three different types: To test if the pS129-aSyn-positive structures observed after challenges were composed of beta-pleated sheets, we performed consecutive immunostaining for pS129-aSyn and Figure 5. Challenges exacerbated motor impairment in a subset of Thy1-Syn14 mice (A) Boxplots illustrating the latency for removal in the different treatment groups (x axis) for WT (red) and TG (blue) mice after 9 weeks. The results shown are for the first paw and the first replicate. TG animals took significantly (Kruskal-Wallis, p < 0.0001) longer than WT animals to remove the tape. On a group-by-group basis, TG mice took significantly longer than WT mice for the FR EC and the FD EC challenge groups. Results were analyzed by Mann-Whitney U test, not corrected for FDR; *p < 0.05; **p < 0.01. Sample sizes: WT FD PBS, n = 4; WT FR DEC, n = 8; WT FR EC, n = 6; WT FD DEC, n = 5; WT FD EC, n = 6; TG FD PBS, n = 4; TG FR DEC, n = 5; TG FR EC, n = 7; TG FD DEC, n = 8; and TG FD EC, n = 7. (B) Summary plot illustrating how the adhesive removal performance evolved from baseline to endpoint in TG mice, plotting the time difference from touch to removal. Only the FD EC group shows a performance drop for three out of six mice. Note: for the TG FR DEC group, only one mouse performed normally and is therefore not representative. Sample sizes: FD PBS, n = 3; FR DEC, n = 1; FR EC, n = 5; FD DEC, n = 6; and FD EC, n = 6. See Table S2 Article ll OPEN ACCESS thioflavin-S (Thio-S) on select sections from challenged TG mice. None of the pS129-aSyn-positive structures ( Figure S7; first two rows) stained positive for Thio-S. This observation was similar to the one we made in the aSyn-preformed fibril injection model ( Figure S7; second row), where intracellular inclusions in the SNpc ( Figure S7; third row) and dorsal striatum (not shown) were also Thio-S negative, despite significant loss of TH-positive neurons in the SNpc and their striatal projections. 86 This indicates that Thio-S-positive inclusions are not a necessary feature of PD-like neurodegeneration. To ensure that our consecutive immuno-/Thio-S staining procedure detects pathologically aggregated protein deposits, we confirmed presence of doubly labeled deposits (amyloid beta and tau) in brain sections of two models of Alzheimer's disease ( Figure S7; bottom two rows). Which form of misfolded or aggregated aSyn is the major pathological culprit in PD is still a matter of debate 87 and beyond the scope of this study.
In summary, it was primarily the EC challenge that increased pS129-aSyn aggregation, a process that was exacerbated by fiber deprivation.
Curli combined with dietary fiber deprivation drove neurodegeneration in the nigrostriatal pathway of Thy1-Syn14 mice The loss of neurons in the SNpc and their projections to the dorsal striatum is a main pathological hallmark of PD. 5 To detect neurodegeneration in TG mice after the different challenges, we stained against tyrosine hydroxylase (TH), an enzyme involved in dopamine synthesis and a marker for dopaminergic neurons and their projections, in both the SNpc and dorsal striatum. Additionally, in the dorsal striatum, we stained for the dopamine transporter (DAT), a marker for dopamine-cycling synapses.
In the dorsal striatum, the combination of FD and EC challenges in TG mice resulted in significantly reduced TH-positive projections when compared to the FR DEC (p = 0.029) and FR EC (p = 0.04) groups ( Figure 6C, top panel). For DAT, we observed a similar pattern ( Figure 6D, top panel). The FD ECchallenged TG mice showed significantly reduced levels in DAT when compared to FD DEC (p = 0.05) and strong trends compared to the FD PBS and FR EC groups ( Figure 6D, top panel).
Quantitation of TH-positive neurons ( Figure 6E, top panel) showed that the exposure to curli caused significant neuronal loss (EC-PBS: FDR = 0.041; EC-DEC: FDR = 1.68EÀ4). FD mice showed an exacerbation of this loss. The difference between the FR EC and FD EC groups showed a strong (p = 0.054) trend, indicating that the FD challenge led to an exacerbation of curli-driven neurodegeneration and no evidence for central, and only minimal peripheral, inflammation in Thy1-Syn14 mice after fiber deprivation and/or curli exposure.
Central, but also peripheral, inflammation is an intrinsic part of PD pathogenesis. 88 To assess inflammation in the brain, we used staining for the microglial marker ionized calcium-binding adapter molecule 1 (Iba1). Surprisingly, we found no evidence for increased Iba1 staining in TG mice of any group compared to their WT or treatment controls ( Figure S8A).
To assess inflammation in the periphery, we measured calprotectin in feces and a panel of chemokines and cytokines in plasma. Calprotectin is secreted into the intestinal lumen by immune and epithelial cells, and it is an inflammation marker for inflammatory bowel disease (IBD) 89,90 or PD. 46, 91 We found a slight but significant increase of calprotectin over time (WT -week 2, p = 0.025; week 6, p = 0.017; post-mortem [PM], p = 0.002; TG -week 1, p = 0.02; week 2, p = 0.003; week 6, p = 0.017; PM, p = 0.059), driven by the FD diet challenge ( Figure S8B). When compared to a colitis model, 92 these values were greatly lower, indicating that, in our model, gut inflammation was minimal.
Patients with PD have increased circulating inflammation markers. 88 Therefore, we tested plasma from a randomly selected subset of mice from each group against a panel of 40 chemo-and cytokines. Surprisingly, TG mice, no matter the challenges, had overall lower inflammation levels than WT mice ( Figure S8C).
We believe these surprising observations can be explained by the probable expression of the human SNCA transgene in hematopoietic cells, known to express Thy1. 93 This could suppress their immune function, a phenomenon observed in other human SNCA transgenic mice. 94 We cannot exclude that a deeper investigation may uncover signs of subtle inflammation in our model. While inflammation certainly plays a role in initiating and driving pathologies in PD and in many models thereof, our result here indicates that, at least in some scenarios, it is not necessary to exacerbate PD-like pathologies. Multi-factor-driven exacerbation of PD pathologies in Thy1-Syn14 mice To obtain an integrated view, parsing out the contribution of each challenge to PD pathology progression, we generated a radar plot. We simplified the output by classifying the results of the different treatment groups from lowest to highest ( Figure 7A). We further split our findings over two categories (brain and gut) and eight sub-categories (gut: alpha diversity decrease, mucus foraging genera, lower gut barrier, lower taxa gut barrier, mucus erosion, and aSyn aggregation (ENS); brain: motor impairment, neurodegeneration, and aSyn aggregation (CNS); Figure 7A).
In TG mice, fiber deprivation was the underlying cause for changes in the gut microbiome and barrier, while bacterial curli drove enhanced aSyn aggregation in both ENS and CNS (Figure 7A). However, overall, the combination of the different challenges (TG FD EC) had the greatest effect on all PD-relevant pathologies ( Figure 7A).
We propose a sequence of events ( Figure 7B), whereby chronic dietary fiber deprivation leads to changes in microbial populations, gut mucus erosion, and increased gut permeability. The resulting higher exposure to amyloidogenic curli causes increased aSyn aggregation in the ENS and CNS, leading to neurodegeneration in the nigrostriatal pathway and exacerbation of motor deficits.

DISCUSSION
While the identification of genetic and environmental factors that modulate PD risk has progressed rapidly, 95,96 less is known about factors that modulate PD progression. 9 Our findings propose a sequence of events, starting with diet-induced dysbiosis, leading to reduced gut barrier integrity, which increases bacterial amyloidogenic protein entry, and ultimately culminating in exacerbation of PD pathologies in ENS and CNS.
The impact of diet on microbial gut health has been widely reported. Rodent studies reported rapid shifts in microbial gut populations after fiber deprivation. 15,16,97,98 Accordingly, our data showed decreased diversity and altered abundances of many PD-associated taxa when mice were fed an FD diet. The lack of dietary fiber makes specialized taxa, which are equipped with glycan-degrading enzymes, such as Akkermansia spp. or the Bacteriodetes genera Alistipes spp. and Bacteroides spp., 65 switch to host glycans as energy resource, promoting mucus erosion and pathogens susceptibility. 16,65,99 Longer-term lack of dietary fiber triggers a compensatory mechanism, increasing mucin production, and re-establishing at least the inner mucus thickness. 15 In our study, only the outer mucus layer showed treatment-induced thinning. The thin outer mucus layer was associated with reduced bacterial diversity, possibly leading to changes in host-microbe interactions. We further observed reduced abundances of the butyrate-producing genera Lachnospiraceaea NK4A136 and Roseburia. Additionally, Lactobacillus, which was also reduced in FD mice, was shown to stimulate butyrate production by such bacteria. 100 Recent studies showed the impact of microbial metabolite changes on gut barrier integrity. The metabolite butyrate, for instance, is essential in regulating energy metabolism, proliferation, and differentiation of gut epithelial cells, has anti-inflammatory properties, stimulates mucin production, and, importantly, promotes gut barrier protection by stimulating expression of ZO-1, ZO-2, cingulin, and occludin. 64,101 Our findings on gut barrier integrity revealed the lowest levels of tight junction proteins ZO-1 and occludin in TG mice challenged by fiber deprivation and curli. This supports the notion that these environmental challenges work in concert with abnormal aSyn to promote a leaky gut barrier. In intestinal biopsies from patients with PD, especially occludin was found at lower levels, whereas ZO-1 was unchanged. 102 Future studies will have to parse out the effect of different environmental challenges on features of gut barrier integrity. Curli is a bacterial protein produced by Enterobacteriaceae. This bacterial family is increased in patients with PD and is associated with disease severity. 103,104 Its role as an aSyn cross-seeding agent, in the promotion of aSyn aggregation and worsening of motor symptoms, was documented in different models, such as C. elegans, 105 Fisher rats, 27 and another line of aSyn-overexpressing mice. 28 Enhanced curli exposure in PD could be the result of its secretion by Enterobacteriaceae for biofilm formation covering the intestinal mucosa. These biofilms could form as a consequence of excessive consumption of antibiotics. 106 Interestingly, excessive and repeated antibiotic consumption leads to enrichment of other PD-relevant taxa such as A. muciniphila, 107 and it is linked to PD risk. 108,109 Either gavaged 27 or supplemented in a human fecal microbiota transplant, 28 curli promotes PD pathologies, such as aSyn aggregation, in gut and brain.
PD initiation and progression are modulated by numerous external factors, for which an important entry point is the gut and from where abnormal aSyn can spread in a prion-like manner via the vagus nerve to the brain. 33, 110 One of the most obvious factors is diet. A ''Western'' diet poor in fiber worsens PD progression, whereas a FR diet delays it. 13 In this study, using an aSyn transgenic mouse model, we show how these factors work together in worsening PD progression. Fiber deprivation shifts the composition of the microbiome toward bacteria that harm gut barrier integrity. Dysbiosis in PD promotes bacteria that produce amyloidogenic proteins. 111 We show that oral administration of E. coli to aSyn-overexpressing mice exacerbates PDlike pathologies, such as phosphorylated aSyn-positive structures and neurodegeneration, which are more pronounced in mice fed an FD diet. Thus, our study indicates that, in the context of initial PD-like disease induced by abnormal aSyn production, PD pathologies are significantly precipitated by environmental factors' action through the gut and its microbiome.
An unexpected observation of our study was the absence of substantial central and peripheral inflammation, which was probably due to aSyn transgene-mediated immunosuppression. Indeed, hematopoietic cells express Thy1. 93 Alpha-synuclein overexpression in isolated microglia and peripheral monocytes greatly reduces, in other aSyn overexpressing mice, the cytokine release by these cells after exposure to lipopolysaccharide. 94 We tentatively conclude that inflammation is not necessary to exacerbate the PD-like pathologies we observed.
In conclusion, we propose a sequence of interacting events involving exogenous and endogenous factors driving PD progression. We underline the importance of a balanced diet in limiting that progression. As their ability to perform activities of daily living declines over time, 112 patients with PD may require special assistance to ensure an optimal diet and oversight of antibiotics' use that could favor the proliferation of curli-producing intestinal bacteria. Our study puts forth the idea of lifestyle adaptations to mitigate PD progression.
Limitations of the study While our study sheds light on interactions between diet, loss of gut barrier integrity, amyloidogenic protein of commensal bacteria, and the manifestations of PD pathologies, the underlying molecular mechanisms of these interactions remain to be uncovered. It is known that bacterial curli cross-seeds with aSyn and thus can initiate a pathological cascade. But how aSyn propagates and which misfolded form(s) of aSyn is/are toxic, in particular in our model after challenges, is unknown. There was no evidence of inflammation in the challenged mice of this study, and the downstream pathological effects of misfolded aSyn also need further investigation. The use of a strong pro-inflammatory challenge, such as LPS or aSyn preformed fibrils, in a study design similar to ours, followed by investigation of mitochondrial, lysosomal, synaptic dysfunctions, and of oxidative and inflammatory stress, may reveal a more complete picture of how PD progression is modulated. Finally, to strengthen the translational relevance of our findings, future studies should include other PD models, ideally in mice of various genetic strain backgrounds that are better models of genetic diversity in human patients.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Paul Wilmes (paul.wilmes@uni.lu).

Materials availability
In this study no new materials have been generated.

Data and code availability
The 16S rRNA gene amplicon sequencing raw data can be accessed at https://www.ebi.ac.uk/ena/browser/home under the accession number PRJEB51988. This paper does not report original code. Access to all other information can be granted upon reasonable request with the lead contact under paul.wilmes@uni.lu.

Ethical Approval
All animal experiments were approved by the Animal Experimentation Ethics Committee of the University of Luxembourg and the appropriate Luxembourg governmental agencies (Ministry of Health and Ministry of Agriculture) and registered under LUPA 2020/ 25. Additionally, all experiments were planned and executed following the 3R guidelines (https://www.nc3rs.org.uk/the-3rs) and the European Union directive 2010/63/EU.

Mice
The transgenic line B6.D2-Tg(Thy1-SNCA)14Pjk 34,116 was used, referred to as Thy1-Syn14 or TG from here on forth. This line overexpresses wild-type human aSyn under the transcriptional regulation of the neuron specific Thy1 promoter. As control animals, wildtype (WT) littermates were used, and all TG were heterozygous for the transgene. All mice used were male, since we observed an unexplained attrition of around 25% of female transgenic mice (untreated) between 4 and 5 months of age. For the characterization of the line, different cohorts were used. For the experimental challenge cohort, 72 male animals, 36 TG and 36 WT littermates, were used. They were singly-caged to avoid coprophagy, had access ad libitum to food and water and were exposed to a regular 12h-daynight cycle. Animals were monitored twice a week. According to welfare guidelines, humane endpoints were set based on different physical parameters, e.g., weight loss/gain, body temperature and coat condition. At the end of the in-life phase, the mice were anesthetized with a mix of 150 mg/kg ketamine + 1 mg/kg medetomidine and subsequently transcardially flush-perfused with 1X PBS. Prior to perfusion, blood was collected from the right atrium. During the in-life phase of the study, 10 mice were either found dead in their home cage or reached a humane endpoint ( Figure S9A). This measure was in line with the animal welfare guidelines.

Experimental design
For the challenge study, 9-months old male mice were randomly assigned to 10 different treatment and respective control groups ( Figure S9A) and treated for a total of 9 weeks; 1-week dietary priming of the colon and additional 8 weeks combined diet and bacterial challenges ( Figure S9B). Food, which was isocaloric (fiber-rich diet: 3.59 kCal/g; fiber-deprived diet: 4 kCal/g), was replaced Article ll every other week and the mice were gavaged with the respective bacteria or sham solution weekly. Body weight and overall health was checked twice a week. Stool samples for microbiome analysis and monitoring of basis gross motor functions via hindlimb clasping and grip strength was also performed weekly ( Figure S9B). After euthanasia, blood, brains and colons for molecular biology and histology were collected.

Bacterial solution preparation and gavage
The E. coli strains used for treatment were C600 (EC) and its isogenic curli-operon knock-out C600:DcsgDEF; DcsgBA (DEC). 25 . Both strains were grown in Lennox broth under aerobic (5% CO 2 ) conditions at 37 C agitating at 300rpm. Bacteria were resuspended in sterile PBS for oral administration at 10 10 CFU/mL They were gavaged with 100mL of bacterial solution, or PBS for the gavage control groups, at a total bacterial load of 10 9 CFUs. Reusable stainless steel 20G feeding needles (Fine Science Tools, 18060-20) were used. Prior and in-between gavages, the feeding needle was washed with filtered 70% ethanol and rinsed with sterile PBS. One group of feeding needles per treatment was used to avoid cross-contamination. Importantly, the mice were not pre-treated with an antibiotic mix because 1) antibiotics have been shown to prevent aSyn aggregation and be neuroprotective, 117 and 2) measuring the impact of the fiber deprivation on a native microbiome was an essential readout.

Tissue collection and preparation
Prior to the transcardial perfusion, up to 400mL of venous blood from the right atrium were collected in EDTA K3 coated collection tubes (41.1504.005, Sarstedt), for plasma cytokine measurements. The tubes were gently inverted and then kept on ice. Plasma was collected after centrifugation at 2000 x g for 10 min, transferred to RNase-free tubes and stored at À80 C.
After perfusion, the brain was placed on ice, and split along the longitudinal fissure into two hemibrains. For molecular biology analyzes, one hemibrain was dissected into different regions of interest (striatum and ventral midbrain). The dissected regions were then put on dry ice and stored at À80 C. The other hemibrains were fixed for immunohistochemistry in 4% PBS-buffered paraformaldehyde (PFA) for 48 h at 4 C and then stored in PBS-azide (0.02%) at 4 C. Then, they were cut to generate 50mm thick free-floating sections using the Leica vibratome VT1000 (Wetzlar, Germany), and sections were stored in a 1% (w/v) PVPP +1:1 (v/v) PBS/ethylene glycol anti-freeze mix at À20 C until staining.
Colon samples for mucus measurements and histopathology were fixed in methacarn (60% absolute methanol: 30% chloroform: 10% glacial acetic acid) solution for 2-4 h, then transferred to 90% ethanol and kept at 4 C. Next, whole colon samples were first transversally cut by hand with a microtome blade into 4-5mm long sections. Those pre-cut sections were then put into a histology cassette while respecting the proximal to distal order. They were held in place in an ethanol-soaked perforated sponge. After 24h post-fixation in 10% formalin, the samples were processed in a vacuum infiltration processor. Finally, all samples were embedded in paraffin, and cut at 3mm on a microtome. If not processed immediately, the slides were stored at 4 C before being stained.
RNA extraction and RT-qPCR for midbrain and striatal aSyn characterization From a separate cohort of 9-months old Thy1-Syn14 mice and corresponding WT littermates, we extracted RNA from dissected colon and different brain regions using the Qiagen RNeasy Plus Universal Mini Kit (Qiagen, 73404). Briefly, 900mL QIAzol lysis buffer (Qiagen, 79306) and three cold 5mm steel balls were added to each sample (previously stored at À80 C). In ice cooled racks, samples were homogenized at 20Hz for 2mins using the Retsch Mixer Mill MM400. Homogenates were transferred to new RNase clean 2mL tubes and left to rest for 5 mins at room temperature (RT). 100mL gDNA eliminator solution was added and the tubes were shaken vigorously for 15 s. Then 180mL of chloroform was added and another strong shake was applied for 15 s. Homogenates were left to incubate for 3 mins at RT. Samples were then centrifuged at 12000 x g for 15 min at 4 C. Five hundred mL of the upper aqueous phase was collected and transferred to new 2mL RNase free tubes. Five hundred mL of ethanol was added to the supernatant and mixed by inverting the tubes back and forth. RNeasy mini spin columns were then loaded with 500mL of the mix, centrifuged at 8000 x g for 30 s at RT, followed by discarding the flow-through from the collection tube. This step was repeated once more. The spin columns were then washed with two different buffers in three steps: one time with 700mL of RWT buffer and twice with 500mL of RPE buffer. At each washing step, the columns were centrifuged at 8000 x g for 30 s at RT, and the flow-through was discarded. Columns were then transferred to new collection tubes and spun at maximum speed for 1min. Finally, columns were transferred to an RNase free 1.5mL Eppendorf tubes. Fifty mL of RNase-free water was added to the columns to elute total RNA. RNA purity and quantity were checked by spectrophotometry using the NanoDrop 2000 (ThermoFisher Scientific) and the Agilent 2100 Bioanalyzer, respectively. Finally, RNA samples were stored at À80 C.
The model used in this study, Thy1-Syn14, carries a transgene for wild-type human aSyn (SNCA). To determine the levels of transcript expression in comparison to endogenous murine aSyn (Snca), quantitative RT-PCR was performed on a separate untreated agematched male cohort (N = 15), using the following primer pairs: Snca For the reverse transcription of RNA to cDNA the SuperScript III RT reverse transcriptase from Invitrogen was used. Briefly, 1mL of oligo (dT) 20 (50mM) and 1mL of 10mM dNTP mix was added to 1mg of total RNA. If needed, nuclease free water was added to obtain the final reaction volume of 13mL. The mixture was briefly centrifuged for 2-3s, heated at 65 C for 5 min, and again chilled on ice for at least 1 min. Another mixture of 4mL 53 first strand buffer, 1mL RNaseOUT (RNase inhibitor), 1mL of 0.1M DTT, and 1mL of Superscript reverse transcriptase (200 U/mL), was added. The final mixture was briefly centrifuged and incubated at 50 C for 1h followed by 15 mins at 70 C for enzyme deactivation. 80mL of RNase free water were added to the reaction mixture. The obtained cDNA was then placed on ice for immediate use, or stored at À20 C for future use.
The qPCR reaction mix contained 2mL of cDNA, 10mM forward and reverse primers, 1X iQ SYBR Green Supermix (Bio-Rad) and PCR grade water up to a volume of 20mL. Each qPCR reaction was run in duplicates on a LightCycler 480 II (Roche). The thermo cycling profile included an initial denaturation of 3 min at 95 C, followed by 40 cycles at 95 C for 30 s, 62 C (annealing) for 30 s and 72 C (elongation) for 30 s, with fluorescent data collection during the annealing step. Data acquisition was performed by LightCycler 480 Software (version 1.5.0.39).

Protein extraction and western blot
For the mouse line characterization, where we used a separate cohort of 9-months old Thy1-Syn14 mice and corresponding WT littermates, total protein was extracted as described before 118,119 with minor adaptations. Briefly, fresh frozen (stored at À80 C) dissected dorsal striatal and midbrain tissue was homogenized in a lysis buffer solution using a potter (Potter-Elvehjem PTFE pestle and glass tube, Sigma-Aldrich, P7734). The lysis buffer volumes were adapted based on sample size (midbrain: 400mL; dorsal striatum: 800mL). For protein extractions from colon samples of our treatment cohort mice, we used fresh-frozen samples and mechanically disrupted them with a potter (Potter-Elvehjem PTFE pestle and glass tube, Sigma-Aldrich, P7734) and homogenized for 30s with an electric pestle in 200mL of 0.5% SDS in PBS. Samples were spun at 750G for 15minutes to remove cellular debris.
All protein supernatants were dosed using Bradford's method assay (Biorad, 5000006). All images were captured using the LI-COR Bioscience C-Digit Chemoluminescence scanner. Subsequent analysis was performed on ImageJ.
Microbial DNA extraction and 16S rRNA gene amplicon sequencing For microbial DNA extraction from single fecal pellets, an adapted version of the IHMS protocol H 120 was used. Fecal samples were preserved in 200mL of a glycerol (20%) in PBS solution and stored at À80 C. Prior to the extraction, the samples were slightly thawed and added 250mL guanidine thiocyanate and 40mL N-lauryl sarcosine (10%). The samples were then left at RT to fully thaw. Then, 500mL N-lauryl sarcosine (5%) were added before the fecal pellet was scattered and vortexed to homogeneity. Samples were then shortly spun down and incubated at 70 C for 1h. Seven hundred fifty mL pasteurized zirconium beads were added to the tubes, then put in pre-cooled racks and horizontally shaken for 7.5 mins at 25Hz in a Retsch mixer mill MM400. Fifteen mg polyvinylpyrrolidone (PVPP) was added and vortexed until dissolved. Then the samples were centrifuged at 20814 x g for 3mins. The supernatants were transferred to new 2mL tubes and kept on ice. The pellet was washed with 500mLTENP (Tris, EDTA, NaCl and PVPP) and centrifuged at 20814 x g for 3mins. This step was repeated three times in total, and each supernatant was added to the previously new 2mL tube. To minimize carryover, the tubes were centrifuged again at 20814 x g for 5mins, and the supernatant was split equally in two new 2mL tubes. One mL isopropanol (Merck) was added to each tube, and mixed in by inverting the tubes. After a 10min incubation at RT, the samples were centrifuged at 20814 x g for 15mins. The supernatant was discarded, and the remaining pellet air dried under the fume hood for 10mins. The pellet was then resuspended in 450mL phosphate buffer and 50mL potassium acetate by pipetting up and down, before the duplicates were pooled and incubated on ice for 90mins. Then, the sample was centrifuged (20814 x g) at 4 C for 35mins, the supernatant transferred into a new tube. Next, 2mL of RNase (10 mg/ml) were added. Then, the tube was vortexed, briefly centrifuged, and finally incubated at 37 C for 30mins. Then, 50mL of sodium acetate, and 1mL of ice-cold 100% ethanol (Merck) were added, and mixed in by inverting the tube several times. The sample was again incubated at RT for 5mins, and Cell Reports 42, 113071, September 26, 2023 23 Article ll OPEN ACCESS centrifuged at 20814 x g for 7.5min. The supernatant was discarded, and the newly formed pellet was subsequently washed three times with 70% ethanol (Merck), and centrifuged at 20814 x g for 5mins. The supernatant was discarded each time. Finally, the clean pellet was dried at 37 C for 15 min, then resuspended in 100 mL TE Buffer, and homogenized by pipetting. After incubation at 4 C overnight, DNA quality and quantity were checked by Nanodrop 2000/2000c and Qubit 2.0 fluorometer (Thermo Fischer Scientific). Samples were stored at À80 C until sequencing.
Five ng of isolated gDNA was used for PCR amplification using primers (515F (GTGBCAGCMGCCGCGGTAA) and 805R (GACTACHVGGGTATCTAATCC)) specific to the V4 region of the 16S rRNA gene. For the first round of PCR, samples were amplified for 15 cycles to avoid over-amplification. Six additional PCR amplification cycles were performed in the second round to introduce sample specific barcode information. All samples were pooled in equimolar concentration for sequencing. Sample preparation and sequencing were performed at the Luxembourg Center for Systems Biomedicine (LCSB) Sequencing platform using v3 2x300 nucleotide paired end sequencing kit for MiSeq.
Sample sizes per week: 16S rRNA gene amplicon sequence analysis Sequence analysis Amplicon Sequence Variants (ASVs) were inferred from 16S rRNA gene amplicon reads using the dada2 package 121 following the paired-end big data workflow (https://benjjneb.github.io/dada2/bigdata_paired.html, accessed: September, 2020), with the following parameters: truncLen = 280 for forward, 250 for reverse reads, maxEE = 3, truncQ = 7, and trimLeft = 23 for forward, 21 for reverse reads. The reference used for taxonomic assignment was version 138 of the SILVA database (https://www.arb-silva. de). 122 Microbial diversity and related statistics Microbiome count data was managed using the phyloseq R package. 123 This package was also used to calculate the Shannon index for alpha diversity and the Bray-Curtis dissimilarity based non-metric multidimensional scaling (NMDS) ordination for beta diversity. Statistical significances of alpha diversity differences were evaluated with the Kruskal-Wallis test (overall comparison between all groups) and the Wilcoxon Rank-Sum Test with false discovery rate correction for multiple comparisons (pairwise contrasts). For beta diversity comparisons, the adonis PERMANOVA test from the R package vegan (Oksanen et al., 2020) 124 was used. All diversity comparisons were performed using ASV count data rarefied to the lowest number of sequences in a sample. Taxon-specific plots (genus and family level) were made using relative abundances (% of taxa out of total).

E. coli quantitative Real-Time PCR (qRT-PCR)
DNA extracted from the mice at weeks 0 (Baseline), 1, 2, 5, 6 and 9, were subjected to qRT-PCR to determine the presence and abundance of E. coli. To achieve this, specific primer-probe sets were designed based on a priori knowledge of target sequences for the gavaged E. coli wildtype strain and a universal 16S rRNA gene. The sequences and the respective primer-probe sets are listed in the key resource table. GBlocks from IDT were used as positive controls, along with DNA extracted from the E. coli strain C600 that was used for gavaging the mice. Negative controls were established using DNA extracted from Fusobacterium nucleatum and Clostridium sciendens. No template controls were additionally used to assess any noise from primer-dimers. The levels of E. coli were obtained by normalised Cq value based on the universal primer-probe set to account for the total bacterial load within each mouse.

Calprotectin ELISA
We measured calprotectin using the S100A8/S100A9 ELISA kit (#K6936, Immundiagnostik AG, Germany). In brief, fecal samples, collected at week 1, 2, 6 and 9, as well as fecal matter from the proximal colon after the mice were sacrificed, were homogenized 1:10 (w (mg)/v (mL)) in extraction buffer by vortexing and subsequently centrifuged for 10 min at 3000 3 g. We transferred the supernatant to a new microcentrifuge tube and used 100mL for ELISA assay. We then followed the protocol as per the provider. The data was analyzed using the 4-parameter algorithm.